Fig. 4: Intracellular PRAF8 partially associates with the Golgi, TGN/EE, and endosomes.

a PRAF8-YFP associates with the endosomes. Top panel: confocal images of PRAF8-YFP (green) in cotyledon epidermal cells stained with the endocytic tracer marker, 8 μM FM4-64 (red) for 40-min (step 1). White circles: overlapping endosomal signals. Middle, step 2, following FM4-64 staining, seedlings were treated with 50 μM cycloheximide (CHX, a protein synthesis inhibitor) for 90-min, followed by the addition of the Arf GEF inhibitor, 70 μM Brefeldin A (BFA), for 60-min. Step 3 (bottom), wash-out with water for 2 h. Yellow arrowheads mark the formation of “BFA-bodies” that overlap with PRAF8-YFP-positive aggregates. Data represent the results of three independent experiments. b–c. Co-localization of YFP/mRFP-PRAF8 (green) with mCherry/YFP-tagged endomembrane markers (magenta) in N. benthamiana leaf epidermal cells. Data represent results of three independent experiments. The co-localization rates (PCC, Pearson correlation coefficient values) are specified at the upper-right corner (cyan). G Golgi, E endosome, TGN/EE trans-Golgi network/early endosome, LE/PVC late endosome/prevacuolar compartment. d Quantification of protein co-localization based on PCC values in (b) and (c). For each pair of co-expression, n > 100 regions-of-interest (ROIs, each 228.3 µm²) were selected to obtain PCC values. ST showed 61/100 ROIs with PCC > 0 (population a), and VAMP721 showed 18/100 ROIs with PCC > 0 (population a). Box plots show the first and third quartile (box), median (line), and mean (cross). Data represent the results of three independent experiments. (z), z-stacked confocal image. Scale bars are 10 μm (a) and 20 μm (b and c).